Cryo-EM Structures Reveal Transcription Initiation Steps by Yeast Mitochondrial RNA Polymerase

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Non-coding 7S RNA inhibits transcription via mitochondrial RNA polymerase dimerization

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The human mitochondrial genome contains a second light strand promoter

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The intercalating beta-hairpin of T7 RNA polymerase plays a role in promoter DNA melting and in stabilizing the melted DNA for efficient RNA synthesis

Phage T7 RNA polymerase contains within its single polypeptide all the elements for specific recognition and melting of its promoter DNA. Crystallographic studies indicate that a beta-hairpin (230-245) with an intercalating valine residue plays a role in promoter opening. We mutated V237 to several amino acids, deleted five amino acid residues at the tip of the hairpin, and mutated E242 and D240 at the base of the hairpin to define the roles of the tip and base of the hairpin in DNA strand separation. The affinity of the hairpin mutants for the promoter DNA was not significantly affected. Stopped-flow kinetic studies showed that the bimolecular rate of DNA binding and the observed rate of pre-initiation open complex formation that corresponds to the sum of DNA opening and closing steps were within 20 to 40 % of the wild-type polymerase. Yet, most mutants showed a smaller amount of the pre-initiation open complex at equilibrium, indicating that the individual rates of promoter opening and closing steps were altered in the mutants. The base mutants, E242A and D240A, showed both a lower rate of promoter opening and a higher rate of promoter closing, suggesting their role in stabilization of the open complex. The V237D and the deletion mutant showed mainly a lower rate of promoter opening, suggesting that the tip of the hairpin may nucleate DNA opening. The defect in pre-initiation open complex formation affected downstream steps such as the rate of the first phosphodiester bond formation step, but did not affect significantly the apparent K(d) of initiating GTPs. We propose that D240 and E242 anchor the hairpin to the DNA and position the tip of the hairpin to allow V237 to intercalate and distort the DNA during open complex formation. The interactions of E242 and D240 with the upstream junction of the melted dsDNA promoter also align the template strand within the active site for efficient RNA synthesis.     

The response of mammalian cells to double-stranded RNA

Double-stranded RNA (dsRNA) has long been recognized as a central component of the interferon (IFN) system. It was originally characterized as a key mediator of IFN induction in response to virus infection. Subsequently, it was identified as a prime activator of the antiviral response. In recent years the discovery of the RNA interference (RNAi) pathway in mammals has renewed interest in dsRNA-mediated cellular responses. This has coincided with the identification of key components of the IFN induction pathway. Here, we present an overview of the current knowledge of dsRNA-mediated pathways in mammalian cells and introduce a link between these pathways and application of RNAi.     

The specificity in promoter recognition by the Bacillus subtilis RNA polymerase deltaA and deltaH holoenzymes is connected with periodicities in the promoter nucleotide sequences

Promoters recognized by deltaA-RNA and deltaH-RNA polymerase have different periodic patterns their nucleotide disposition. By using the special method of Fourier analysis for symbolic sequences. Fourier spectra of the primary structure of promoters transcribed bv deltaA-RNA and deltaH-RNA polymerase were obtained. For two data sets, a small one and a big one, a stepwise discriminant analysis with jackknife test based on the spectral characteristics of each of the promoters was performed. It was shown that it is possible to classify the data with the accuracy of 100% sets into classes: promoters that are recognized by the deltaA-RNA polymerise and promoters that are recognized by the deltaH-RNA polymerase. Significant correlations between the promoter strength and the characteristics of their Fourier spectra were obtained. Thus, the periodicity in nucleotide distribution along the polynucleotide chain is the attribute sufficient for promoter recognition by RNA polymerase holoenzyme.     

Macrolide- and tetracycline-adjustable siRNA-mediated gene silencing in mammalian cells using polymerase II-dependent promoter derivatives

RNA interference has emerged as a powerful technology for downregulation of specific genes in cells and animals. We have pioneered macrolide- and tetracycline-adjustable short interfering RNA (siRNA) expression for conditional target gene translation fine-tuning in mammalian/human cell lines based on modified RNA polymerase II promoters. Established macrolide- and tetracycline-dependent transactivators/trans-silencers bound and activated modified target promoters tailored for optimal siRNA expression in response to clinical antibiotics' dosing regimes and modulated desired target genes in Chinese hamster ovary (CHO-K1) and human fibrosarcoma (HT-1080) cells with high precision. Further optimization of adjustable RNA polymerase II-based siRNA-specific promoters as well as their combination with various transmodulators enabled near-perfect regulation configurations in specific cell types. Devoid of major genetic constraints compared to basic RNA polymerase III-based siRNA-specific promoters, we expect RNA polymerase II counterparts to significantly advance siRNA-based molecular interventions in biopharmaceutical manufacturing and gene-function analysis as well as gene therapy and tissue engineering.